Process for the preparation of inoculum for use in the fermentative production of sodium gluconate



Patented Apr. 29, 1952 QFFECE PROCESS FOR THE PREPARATZUN 0F IN OCULUMFOR USE IN THE FERMENTATIV E PRODUOTIGN OF SODIUM GLUCONATE Russell H.Eleni, Peoria Heights, and Virgil 1E.

8011115 and Andrew J. Moyer, Peoria, 111., assignors to the UnitedStates of America as represented by the Secretary of Agriculture NoDrawing. Application December 29, 1949, Serial No. 135,852

(Granted under the act of March 3, 1883, as amended April 30, 1928; 3'700. G. 757) 3 Claims.

This application is made under the act of March 3, 1883, as amended bythe act of April 30, 1928, and the invention herein described, ifpatented in any country, may be manufactured and used by or for theGovernment of the United States of America for governmental purposesthroughout the world without the payment to us of any royalty thereon.

This invention relates to the production of sodium gluconate byfermentation, and relates more particularly to the preparation of aninoculum with which fermentation media may be inoculated. It has amongits objects the provision of a method for reducing the time and labor inthe production of sodium gluconateby avoiding the direct use of sporesas a means of inoculation. Another object is the preparation. of agerminated inoculum of gluconic acid-producing organisms particularlymold organisms adapted for the fermentative production of sodiumgluconate.

The fermentative production of sodium gluconate, as described andclaimed in the oopending application of Crocker, Moyer and Pfeifer,Serial No. 137,901 filed January 10, 1950 involves the use of sodiumbases as a means for controlling the hydrogen ion concentration of thefermenting media.

The fermentations may be inoculated in a variety of ways. For example,sporulated. surface cultures of the gluconic acid-producing organism maybe prepared, the spores suspended in water, and this suspension ofspores added to the sterile medium. Alternatively, in the course ofproduction the filtered mycelium from previous fermentation may beemployed to inoculate subsequent media.

Both of these two methods possess practical disadvantages which addgreatly to the cost of the sodium'gluconate. If suspension of spores areemployed, a large quantity is necessary. The preparation of the sporesuspensions is tedious, requires a large number of individual flasks ofspores and is time-consuming. Moreover, the necessary incubation periodfor the spores decreases considerably the capacity of the productionfermentation equipment. The second meth- 0d, 1. e., re-use of thefiltered mycelium eliminates some of the difficulty by avoiding theincubation periods and the use of spore suspensions for eachfermentation, but is also accompanied by practical difiiculties andexpense of handling the filtered mycelium. Furthermore, the myceliumcannot be re-used indefinitely, and periodic inoculation of productionbaths with spore suspensions is therefore necessary.

A further alternative for inoculating produc tion fermentations is theuse of a special medium in Which is produced a germinated inoculum.

The special medium is inoculated with a suspension. of spores andincubated under conditions which induce germination of the spores. Thisalternative has been described in connection with the production ofcalcium gluconate (Moyer et al., Ind. and Eng.-Chem.-29,777; Wells etal., ibid. 653), and has been employed in the gallic-tannic acidfermentation and in penicillin production. The use of a germinatedinoculum possesses many advantages, a major one being markedly decreasedinitial lag period in the production fermentation.

Gluconic acid-producing fermentations by molds differ from many otherfermentations in that the activity of the organisms, i. e., the abilityto produce gluconic acid is impaired by ,excessive acidity. If theacidity occurs in the inoculum, the detrimental effect extends to thesubsequent production fermentation. Thus, if free gluconic acid ispresent in sufficient amounts in either the inoculum or the productionmedium, the rate of further acid production will approach zero. Thisphenomenon is caused, it is believed, by inactivation of the oxidativeenzyme system of the mold by exposure of the organism or the enzymes toexcessive acidic conditions.

In the Work of Moyer et al., previously noted, it was disclosed that thepH of the medium used for germination can be controlled by the additionof calcium carbonate. For many purposes, however, including theproduction of sodium gluconate by direct fermentation, the presence ofcalcium ions in the medium is detrimental.

It is also indicated in the Moyer publication that satisfactory inoculumcan be prepared without the use of calcium carbonate if the supply ofoxygen is limited. However, in this previous work a high concentrationof spores per unit volume of germination medium was employed. Underthese conditions, employing shaker flasks, a satisfactory inoculum wasprepared in which the pH did not fall below 4.4 in a medium containingno added buffering agency. A slight reduction in the number ofungerminated spores per unit volume of germination medium was madeployed as a satisfactory inoculum and is fully as efiective aspreviously known inoculant media. Applicants have discovered that oneunit weight of mycelium grown from agiven number of spores underconditions permitting great mycelial growth per spore is equivalent inglu conic acid-producing capacity to the same weight of mycelium of theprior art, obtained. from a relatively limited growth of say, 30 timesas many spores. Utilizing their discovery, applicants provide a processfor preparing a germinated inoculum which permits a great saving in thenetnumber of spores required to conduct the duction fermentation.

The present invention proa reduction in the required number ofungerminated spores, far beyond that previously known. The preparationand handling of seed flasks .is a highly technical and costly operation,and applicants process reduces the required number of spores toone-thirtieth of the amount required by the best previously knownmethods. When large-scale fermentations are considered, this economy inspores is reflected in a greatly reduced number of seed flasks requiredand a substantial saving in cost and labor.

We have found that the mycelial growth per spore is large whengermination and growth occurs in the absence of an added bufferingagency and under suitable nutrient conditions and conditions ofsubmerged aeration with limited oxygen supply, and, moreover, that thislarge mycelial growth may be used as a satisfactory inoculum if the timeof incubation and the oxygen supply are so balanced that the pH of thegermination medium remains above 4.5. The oxygen supply can becontrolled by varying the air pressure used in aeration, by varying. theintensity of mechanical stirring, if any is employed, and by varying thevolume of air blown. through the medium. The time of incubation.

which should approximate 24 hours for convenience, may be shortened orlengthened within a fairly wide range. Balancing the time and rate ofoxygen supply is a fairly simple matter since the acidity increases, i.e., the pH value decreases, with increased oxygen and. increased. time.

For our own purposes. we arbitrarily setthe time for incubation atapproximately 20 to 30 hours. This time range isspecified herein, sinceit represents a convenient mode ofpracticing, the.

invention. It is to be understood that other predetermined times may beemployed or alternatively one may hold constant at a predetermined valuethe rate of oxygen supply.

Employing conventional equipment we have found that, with air as thesource of oxygen, at atmospheric pressure, the pH'will be maintainedwithin the desired range at aeration rates of 0.1 to 0.35 volume of airper volume of medium per minute. The greater rate of aeration will becorrelated with a correspondingly lesser time. and.

vice versa. With the timerange arbitrarily fixed at 20 to 30 hours thepH of the mediumcan. be controlled by careful regulation of the air so.that the spores germinate and grow rapidly to produce is thereforedistin-- guished from prior processes in that it permits.

a highly active inoculum. In contrast to what would be expected, the pHof the final germinated medium can be maintained above a value onlyslightly less than theinitial value, and it is still within the rangesuitable for good vegetative growth of the gluconic acid-producing moldorganisms.

If a surplus of air is provided, the pH decreases at a rate'which bringsit to a value injurious to the enzymesystem of the organism and to theability of the organisms to produce the enzymes needed for thesubsequent conversion of glucose to gluconic'acid. The critical pH atwhich inactivation begins: to occur appears to be about 4.0 to 4.5. If adeficiency of air is provided, poor germination of spores and poorgrowth of mycelium may result.

In an analogous manner, as previously noted, the time. may be variedwith any given rate of oxygen supply, but in general the rate of oxygensupply is relatively low. As germination proceeds, the pH falls slowly,and the germinated medium is ready for use as an inoculum when thelimiting value of 4.5 is reached.

The nutrient medium we employ is characterized by a relatively lowcarbohydrate level, of the order of 3 to 12 percent. Germinationtemperatures for our process fall within the range-of 20 to 35 C. Theseconditions are typical of germi nation media relating to gluconic acidfermentation processes.

The following experimental data illustrates our invention.

Fifty gallons of germination medium of the following composition wasprepared and sterilized in a closed tank equipped with a sparger for theintroduction of air.

TABLEI Composition of inoculum medium Percent Glucose 5.0 Corn steepliquor (as received) 0 .5: MgSO4-7H2O 0.02 KH2PO4 0.02

Urea 0.01 (NI-I4) 2HPO4 0.04:1

After the nutrient solution was cooled, it was inoculated with asuspension of spores of 'A'speh gillus niger and the medium wasaerated'at-therate of 0.2 volume of air per volume of medium per minute.At the end of 24 hours, an excellent growth of vegetative cells wasobtained. Three separate experiments were carried out, andthepH of themedium decreasedonly slightlyduring: the incubation period as is shownby the following table.

TABLE II pH of medium Run No. Initial 2x The criticalpHiat whichinactivation of the enzyme system begins appears to be about.4'.0i to4.5. Our process therefore produces an ino.c-. ulum which has not beeninjured by subjection to conditions of too high acidity. Thetutilityofthe inocula is, illustrated" by thefollowing pilot'- plantdata.- Thecomposition of thesodium: gluconate production medium afterinoculationwith varying amounts of our germinated inoculum was asfollows:

TABLE III Composition of production medium Percent Commercial glucose23.5 Corn steep liquor (as received) 0.44 MgSO4-7H2O 0.02 KHzPOtl 0.02

Urea 0.01 (NHt) 2HPO'4 0.04

The fermentations were considered complete when the residual sugarcontent of the medium was 0.2 percent or less. The results 'of thefermentations are tabulated below.

We have found that percent by volume of our inoculum is sufficient 'forfast and complete fermentation of glucose to gluconic acid, in the formof sodium gluconate. Since this quantity of culture was produced fromthe equivalent of 66 sq. cm. of sporulated surface cultures, it followsthat only 20 flasks of spores would be required to produce inoculum fora commercial scale fermentation of 5000-gallon batches. This compareswith 327 flasks when the germinated spores, prepared by the method ofthe literature references referred to above, are used; or 1000 flaskswhen spores are employed directly as the inoculum.

We claim:

1. A method for the production of a germinated inoculum of gluconicacid-producing mold organisms suitable for inoculating sodium gluconatefermentation media which comprises inoculating a nutrient 'mediumcomprising 3 to 12 percent glucose with spores of a gluconicacid-producing mold, incubating the nutrient medium under conditions ofsubmerged aeration until suflicient growth of vegetative cells isobtained, and maintaining the pH of the medium above 4.5 by controllingthe rate of aeration.

2. Process according to claim 1 in which the rate of, aeration isbetween 0.1 and 0.35 volume of air per volume of medium per minute.

3. A method for the production of a germinated inoculumof Aspergz'llusnzger which comprises inoculating a nutrient medium comprising 3 to 12percent glucose with spores of Aspergillus niger, incubating thenutrient medium for a period of 20 to 30 hours under conditions ofsubmerged aeration and maintaining the pH of the medium above 4.5 bycontrolling the rate of aeration.

RUSSELL H. BLOM. VIRGIL E. SOHNS. ANDREW J. MOYER.

REFERENCES CITED.

The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 2,277,716 Lockwood Mar. 21, 19422,351,500 Mayer June 13, 1944 2,454,753 Hager Nov. 23, 1948 OTHERREFERENCES Moyer et al., Ind. & Eng. Chem, July 1937, pp. 777-781.

Wells et al., Ind. & Eng. Chem, June 1937, pp. 653-656.

1. A METHOD FOR THE PRODUCTION OF A GERMINATED INOCULUM OF GLUCONICACID-PRODUCING MOLD ORGANISMS SUITABLE FOR INOCULATING SODIUM GLUCONATEGERMENTATION MEDIA WHICH COMPRISES INOCULATING A NUTRIENT MEDIUMCOMPRISING 3 TO 12 PERCENT GLUCOSE WITH SPORES OF A GLUCONICACID-PRODUCING MOLD, INCUBATING THE NUTRIENT MEDIUM UNDER CONDITIONS OFSUBMERGED AERATION UNTIL SUFFICIENT GROWTH OF VEGETATIVE CELLS ISOBTAINED, AND MAINTAINING THE PH OF THE MEDIUM ABOVE 4.5 BY CONTROLLINGTHE RATE OF AERATION.